Purpose: :
PKC dependent signaling pathway is well known regulator of Matrixmetalloproteinases (MMPs) which play an important role duringdevelopment. The goal of this study is to investigate whetherinjection of staurosporine (STS) into the early developing chickembryos in ovo affects the differentiation of retinal neuroblastsduring retinal development, and is to compare whether inhibitionof MMP activities differentially regulates the STS-induced neuronalcell differentiation and cell outgrowth in both coating andnon-coating culture conditions.

Methods: :
Neuroblasts isolated from an early stage of developing chickretinas at embryonic day 6 days (E6) were primary cultured incoating culture dishes with poly DL-ornithine and also non-coatingdishes for 3 days. Neuroblasts were treated with STS in thepresence of GM6001 (broad MMPs inhibitor), GI254023X (ADAM10specific inhibitor), and amiloride (uPA inhibitor). Furthermore,to investigate the differentiation pattern of neuroblasts inovo, STS and/or amiloride were directly injected into developingeggs at E6. Two, four, and six days after injections, eye ballswere harvested and embedded in OCT. Indirect immunofluorescencestaining for Tuj-1, visinin, neurofilament, CD44v6, and N-cadherinin cultured cells and frozen sectioned retinas was performed,and their expression levels were also examined by Western blotanalysis.

Results: :
In both culture conditions, STS induced morphological changesdifferentially and also neurite outgrowth. Cell cycle analysisshowed that STS treated cells were growth arrested at the G2-Mphage. High levels of visinin and neurite outgrowth as wellas cleavage forms of both CD44v6 and N-cadherin induced by STSwere strongly inhibited by GM6001 and amiloride. Furthermore,expression level of visinin induced by STS in photoreceptorcell layer was also dramatically decreased by amiloride in ovo.